Current research on the regulation of G protein-coupled receptors (GPCRs) shows that feedback regulatory loops in the target cell affect the functional properties and density of these receptors in a coordinate fashion. Two important aspects of this regulation are the phosphorylation of the GPCRs by a family of protein kinases known as G protein-coupled receptor kinases (GRKs) and the association of GPCRs with a family of proteins known as arrestins. The complex formed by the phosphorylated GPCR and the arrestins is believed to be a common molecular intermediate in the uncoupling of the GPCRs from G proteins, the internalization of GPCRs and the subsequent recycling and/or down-regulation of the internalized receptors. The follitropin receptor (FSHR) is a unique member of the GPCR family in several aspects. Among these, the uniqueness of loci of phosphorylation (i.e., the first and third intracellular loops) and the non-essential role for phosphorylation on internalization predict an unusual mode of regulation when compared with other GPCRs. The studies proposed herein will further explore how arrestin-2 or -3 affect the uncoupling and the trafficking of the FSHR and will define the structural motifs of the FSHR that are involved in internalization, uncoupling, and arrestin binding. The specific aims are as follows: (1) Test the hypothesis that the GRK2-catalyzed phosphorylation of the FSHR is necessary for internalization while the GRK6-catalyzed phosphorylation is necessary for uncoupling. We also propose that arrestin-2 and arrestin-3 may play different roles in uncoupling and internalization. ) Define the structural features of the FSHR that are responsible for binding arrestin-2 or -3. (3) Define the structural features of the FSHR that are involved in the internalization of follitropin (FSH) and the uncoupling of the rFSHR from its effector system. (4) Define the fate of the internalized FSH-FSHR complex and the functional consequences of internalization. These studies should provide valuable insights about the regulation of the function and the cellular trafficking of the FSHR in particular and GPCRs in general.